The TRAF protein family comprises seven members, i.e. TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6 and TRAF7, which largely display homologies in their domain structures (Xie, P., TRAF molecules in cell signaling and in human diseases. J Mol Signal, 2013. 8(1): p. 7 and Zotti, T., P. Vito, and R. Stilo, The seventh ring: exploring TRAF7 functions. J Cell Physiol, 2012. 227(3): p. 1280-4). Studies of the past years have unraveled a large number of signaling receptor families that embrace TRAF proteins for signal progression. These pathways include Toll-like receptors (TLRs), IL-1 receptor family, T-cell receptor, IL-17 receptors, TNF receptors and others. TRAF-dependent signaling pathways mainly facilitate activation of (i) the transcription factor NF-κB and (ii) mitogen-activated protein kinases (MAPKs), thereby contributing to the control of various cellular processes such as survival, proliferation, differentiation, activation, cytokine production and autophagy (Xie, P., TRAF molecules in cell signaling and in human diseases. J Mol Signal, 2013. 8(1): p. 7). Hence, distinct alterations in TRAF family members have been shown to contribute to the pathogenesis of individual human diseases including autoimmunity, immunodeficiency and cancer (Hildebrand, J. M., Z. Yi, C. M. Buchta, et al., Roles of tumor necrosis factor receptor associated factor 3 (TRAF3) and TRAF5 in immune cell functions. Immunol Rev, 2011. 244(1): p. 55-74; Namjou, B., C. B. Choi, I. T. Harley, et al., Evaluation of TRAF6 in a large multiancestral lupus cohort. Arthritis Rheum, 2012. 64(6): p. 1960-9 and Netea, M. G., C. Wijmenga, and L. A. O'Neill, Genetic variation in Toll-like receptors and disease susceptibility. Nat Immunol, 2012. 13(6): p. 535-42).
The TRAF6 protein functions both as an adaptor protein as well as an E3 ubiquitin ligase to activate the transcription factor NF-κB. In particular, TRAF6 exhibits a RING domain and four Zincfinger domains at its N-terminus, whereas the C-terminal TRAF domain contains a Coiled-Coil and a conserved MATH domain (Yin, Q., S. C. Lin, B. Lamothe, et al., E2 interaction and dimerization in the crystal structure of TRAF6. Nat Struct Mol Biol, 2009. 16(6): p. 658-66). TRAF6 directly interacts with the heterodimeric E2 enzyme Ubc13/Uev1a with its N-terminus (RING and Zincfinger1) to attach K63-linked ubiquitin chains to its substrate proteins. This process is critical for progression of signal transduction in several cellular signaling processes like T-cell receptor mediated NF-κB activation (Oeckinghaus, A., E. Wegener, V. Welteke, et al., Malt1 ubiquitination triggers NF-kappaB signaling upon T-cell activation. EMBO J, 2007. 26(22): p. 4634-45) or innate immune response including IL-1β and Toll-like receptor signaling (Bhoj, V. G. and Z. J. Chen, Ubiquitylation in innate and adaptive immunity. Nature, 2009. 458(7237): p. 430-7). Moreover, TRAF6 is involved in IL-17 receptor signaling as well as in DNA damage response (Xie, P., TRAF molecules in cell signaling and in human diseases. J Mol Signal, 2013. 8(1): p. 7 and Walsh, M. C., J. Lee, and Y. Choi, Tumor necrosis factor receptor-associated factor 6 (TRAF6) regulation of development, function, and homeostasis of the immune system. Immunol Rev, 2015. 266(1): p. 72-92). Overexpression of TRAF6 as well as enhanced TRAF6 activity have been shown to promote chronic immune stimulation and cytokine secretion, thereby causing a broad variety of disorders including autoimmune diseases, inflammation and cancer. Taken together, targeting TRAF6 activity by disrupting the TRAF6-Ubc13 binding displays a very attractive novel strategy to counteract disease formation.
Previous studies have mainly focused on inhibiting the protein-protein interaction (PPI) between the E3 ligase and its substrate in order to diminish ubiquitination; with p53-Mdm2 being the most prominent example (Vassilev, L. T., B. T. Vu, B. Graves, et al., In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science, 2004. 303(5659): p. 844-8). International patent application WO 2011/160016 A2 relates to an isolated protein fragment that includes a binding pocket or active site on an E3 ligase that modulates the E2-E3 interface, and to an agent that interacts with such a binding pocket. WO 2011/160016 A2, however, only mentions two agents denoted as CRIN-1 and CRIN-2 for such purpose.
Thus, there is a great need for new, first-in-class inhibitor scaffolds which can inhibit TRAF6, in particular TRAF6 E3 ligase activity by disrupting its interaction to the E2 enzyme Ubc13.
The inventors of the present invention have conducted intensive studies and found surprisingly, that the compounds according to Formula I, Formula II, Formula III and Formula IV, which are described in more detail below, satisfy this need. Without wishing to be bound by theory, the inventors believe that the compounds according to the present invention target the protein-protein interaction of TRAF6 (E3 ligase)-Ubc13 (E2 enzyme) and thereby interrupt this biological pathway (E2-E3 inhibitors). In particular, it is believed that the compounds counteract the catalytic activity of TRAF6 and thereby leading to reduced NF-κB activation.
The compounds according to the present invention are effective in reducing NF-κB activation in cell lines (see FIG. 4 and FIG. 6) as well as in primary T cells obtained from BALBc mice (see FIG. 8). Further, it was found by the inventors that the compounds according to the present invention ameliorate Rheumatoid Arthritis disease outcome in a CIA mouse model (see FIG. 13), ameliorate Psoriasis disease outcome in an IMQ-induced psoriasis mouse model (see FIG. 12), selectively kill ABC-DLBCL with chronic MYD88 signaling (see FIG. 14), reduce gain of weight in a T2D mouse model and additionally reduce IL-1β expression (see FIG. 11), reduce proliferation of U2OS cells to the same degree as 2Gy irradiation and application of the compounds together with irradiation leads to synergistic effects (see FIG. 15), and the compounds affect immune receptor signaling in primary human peripheral blood mononuclear cells (see FIG. 7). On a molecular basis, without wishing to be bound by theory, it is believed that the compounds of the present application disrupt TRAF6-Ubc13 binding (see Discussion).